Calmodulin interacts with Rab3D and modulates osteoclastic bone resorption

Abstract

Calmodulin is a highly versatile protein that regulates intracellular calcium homeostasis and is involved in a variety of cellular functions including cardiac excitability, synaptic plasticity and signaling transduction. During osteoclastic bone resorption, calmodulin has been reported to concentrate at the ruffled border membrane of osteoclasts where it is thought to modulate bone resorption activity in response to calcium. Here we report an interaction between calmodulin and Rab3D, a small exocytic GTPase and established regulator osteoclastic bone resorption. Using yeast two-hybrid screening together with a series of protein-protein interaction studies, we show that calmodulin interacts with Rab3D in a calcium dependent manner. Consistently, expression of a calcium insensitive form of calmodulin (i.e. CaM1234) perturbs calmodulin-Rab3D interaction as monitored by bioluminescence resonance energy transfer (BRET) assays. In osteoclasts, calmodulin and Rab3D are constitutively co-expressed during RANKL-induced osteoclast differentiation, co-occupy plasma membrane fractions by differential gradient sedimentation assay and colocalise in the ruffled border as revealed by confocal microscopy. Further, functional blockade of calmodulin-Rab3D interaction by calmidazolium chloride coincides with an attenuation of osteoclastic bone resorption. Our data imply that calmodulin- Rab3D interaction is required for efficient bone resorption by osteoclasts in vitro.

Figure 1. Calmodulin interacts with Rab3D.

(A) A yeast two hybrid assay showing that Calmodulin interacts with Rab3D, by using histidine-deficient plate. (B) BRET assays showing that co-transfection of Rluc-Camodulin and EYFP-Rab3D fusion protein constructs resulted in a significant BRET signal. Co-expression of Rluc and EYFP is shown as a negative control. (C) Flag-Rab3D proteins expressed in COS cells interact with calmodulin saphorose in the presence of 2 mM calcium. *Indicates p Value < 0.001 when compared with EYFP and Rluc. (D) Calmodulin calcium-insensitive mutant perturbs its interaction with Rab3D. Generation of a Rluc-calmodulin construct in which four aspartic acid residues at position 23, 59, 96, 132 were substituted with alanine, mimicking a calcium insensitive form of calmodulin. (E) BRET assays showing that the calcium insensitive form of camodulin failed to interact with Rab3D. 1:1, 1:2 and 1:3 indicate that transfected plasmid ratio of EYFP-Rab3D/ Rluc-camodulin or EYFP-Rab3D/ Rluc-calmodulin mutant 1234. Symbol *indicates p Value < 0.001 when compared with EYFP and Rluc-camodulin control. Symbol # indicates p Value < 0.001 when compared Rluc-camodulin with Rluc-calmodulin mutant 1234.

Figure 2. The interaction of Calmodulin with Rab3D has a closer proximity when Rab3D is GTP-bound.

(A) Predicted molecular structures of wild-type Rab3D, GTP-bound Rab3D (Rab3DQ81L), nucleotide empty RAB3D (Rab3DN135I) and prenylation motif deletion of Rab3D (Rab3DΔCXC). (B) EYFP fusion protein constructs of EYFP-Rab3D, EYFP-Rab3DQ81L, EYFP-Rab3DN135I and EYFP-Rab3DΔCXC that were used for BRET assays. (C) Western blot analysis showing the expression of EYFP-Rab3D, EYFP-Rab3DQ81L, EYFP-Rab3DN135I and EYFP-Rab3DΔCXC proteins by anti-GFP. (D) BRET assays showing that calmodulin exhibited an enhanced association with a GTP-bound Rab3D (Rab3DQ81L) when compared to wild-type Rab3D, nucleotide empty RAB3D (Rab3DN135I) and prenylation motif deletion of Rab3D (Rab3DΔCXC) in BRET assays. *Indicates p Value < 0.001 when compared with EYFP and Rluc. # indicates p Value < 0.05 when compared to wild-type Rab3D, nucleotide-empty (Rab3DN135I) and prenylation motif deletion of Rab3D (Rab3DΔCXC).

Figure 3. Calmodulin and Rab3D are co-expressed during osteoclast formation and co-fractionated in the membrane fraction of osteoclasts.

(A) Representative images from BMMs cultured in the present of M-CSF and RANKL for a period of 0, 1, 3, 5 days and then fixed and stained for TRACP activity. (B) Semi-quantitative RT-PCR showing the co-expression of calmodulin and Rab3D along with osteoclast marker gene expression of TRACP, NFATc1, DC-STAMP, V-ATPase d2, and calcitonin receptor. (C) Western blot analysis showing that calmodulin and Rab3D proteins are expressed at similar kinetics during osteoclastogenesis. (D) Sucrose gradient sedimentation assays showing that Rab3D and calmodulin co-fractionate in the large membrane faction (P = Pellet). Note that all Western blot results with cropped blots are displayed from well established sizes of proteins; calmodulin, Rab3D and VATPase (d2).

Figure 4. Colocalisation between Rab3D and calmodulin in bone-resorbing BMM-derived osteoclasts.

(A) Representative confocal microscopy images of individual and overlay fluorescent channels of Rab3D (green), calmodulin (red), F-actin (blue) and nuclei (magenta). Colocalisation between Rab3D and calmodulin appears as yellow in overlay. Red line demarcates the ruffled border region within the sealing zone. White line corresponds to the correlative linescan analysis in (B). Bar = 10 μm.

Figure 5. Functional blockade of the interaction of Rab3D and calmodulin by calmidazolium chloride attenuates osteoclastic bone resorption.

(A) BRET assays showing the effect of calmidazolium chloride on the interaction of calmodulin and Rab3D, Rab3DQ81L, Rab3DN135I and Rab3DΔCXC; respectively. (B) Treatment of osteoclasts with calmidazolium chloride inhibits osteoclastic bone resorption with quantitative analysis of bone resorption areas. BMM derived osteoclasts were seeded into bone slices in the presence and absence of calmidazolium chloride for 24 hours. (C) Total number of TRACP positive osteoclastic like cells. (D) Representative images of bone resorption assays showing the effect of calmidazolium on TRACP positive osteoclast morphology (upper panel), and osteoclastic bone resorption SEM images (lower panel). Scale bars are shown. * and ^#Indicate p Value < 0.001, and < 0.05 respectively when compared to untreated control.

Figure 6. A working model illustrating that calmodulin is concentrated on the ruffled border membrane in osteoclasts and mediates bone resorption process.

Rab3D interaction with calmodulin is implicated for a role in bone resorption when Rab3D-bearing vesicles reach the resorbing ruffled border compartment of an osteoclast.